Preventing human immunodeficiency virus (HIV-1) transmission by vaccination is a highly desirable, but elusive, goal. Vaccine-induced antibodies that bind the HIV-1 envelope glycoprotein (Env) spike and neutralize the virus are thought to be a desirable component of a protective immune response; as a consequence, recombinantly expressed immunogens containing the exterior portion of Env are generally regarded as an essential element of a prophylactic vaccine. The HIV-1 Env is highly glycosylated, with glycans accounting for about 50% of the glycoprotein mass. These glycans contribute significantly to a number of viral functions, including the ability of HIV-1 to evade antibody responses. The presence and type of glycans on Env-based vaccine candidates influence both immunogenicity and antigenicity of the protein, and several of the gp120 and gp41 glycans serve as epitopes or components of epitopes targeted by potent HIV-1-neutralizing antibodies. If there is any hope of eliciting these types of antibodies during vaccination, the glycans on the Env vaccine must be of the same type as those found on the virus. Therefore, two critical questions facing the field of HIV-1 vaccine development with respect to Env glycosylation are: (1) What is the glycan profile of native HIV-1 Env? and (2) How can that glycan profile be recapitulated in an Env-based vaccine? Answering these two fundamental questions would represent a significant advance in HIV-1 vaccine research: If the glycan profile of a vaccine candidate could match that of native virus, the chances of eliciting effective antibodies that contain glycan-based epitopes would be increased. The two goals of this application are to characterize the glycosylation of Env from the most native source possible, from T-lymphocytes, and to identify ways to express Env with the correct glycosylation in a more tractable cell line that could be used in GMP manufacturing of Env for human clinical trials. This project will be run by a synergistic, collaborative team wit complementary strengths. The group has recently published the glycosylation profile of an HIV-1 membrane Env trimer, taking advantage of highly relevant technical innovations in Env glycoprotein production, sample preparation, and glycosylation analysis; these recent developments provide a strong foundation for the studies proposed herein. The successful completion of the project will provide all vaccine developers with the tools they need to generate native glycosylation profiles on their Env of choice.